Intermediates in the preparation of secretin

ABSTRACT

THIS INVENTION RELATES TO INTERMEDIATES AND THEIR SALTS USEFUL IN THE PREPARATION OF THE GASTROINTESTINAL HORMONE, SECRETIN. THE INTERMEDIATES OF THIS INVENTION COMPRISE PARTIAL SEQUENCES OF AMINO ACIDS, WHICH, WHEN COMBINED, FORM THE SECRETIN MOLECULE. THE NOVEL PROTECTIVE PEPTIDES CORRESPONDING TO THESE PARTIAL SEQUENCES ARE: (1) PROTECTED L-HISTIDYL-L-SERYL-L-ASPARTGLYGLYCINE; (AMINO ACIDS 1 TO 4); (2) PROTECTED L-THREONYL-L-PHENYLALANYL-L-THREONYL-L SERINE HYDRAZIDE; (AMINO ACIDS 5 TO 8); (3) PROTECTED L-GLUTAMYL-L-LEUCYL-L-SERYL-L-ARGINYL-LLEUCINE HYDRAZIDE; (AMINO ACIDS 9 TO 13); AND (4) PROTECTED L-ARGINYL-L-ASPARTYL-L-SERYL-L-ALANYL-LARGINYL-L-LEUCYL-L-GLUTAMINYL-L -ARGINYL-L-LEUCY-LGLUTAMINYLGLYCYL-L-LEUCYL-L-VALINAMIDE; (AMINO ACIDS 14 TO 27).

INTERMEDIATES IN THE PREPARATION F i SECRETIN Miklos Bodanszky,Princeton, Miguel A. Ondettr, Highland Park, Malcolm H. von Saltza,Millstone, and

Venkata'chala L. Narayanan and Seymour D. Levine,

North Brunswick, N.J., assignors to E. R. Squibb & Sons, Inc'., NewYork, N.Y.

No Drawing. Continuation of application Ser. No.

736,880, Apr. 12, 1968, which is a division of application Ser. No.553,290, May 27, 1966, now Patent No. 3,400,118. This application Oct.20, 1970, Ser. No. 82,510

Int. Cl. C07c 103/52 US. Cl. 260-112.5 Claims ABSTRACT OF THE DISCLOSUREThis invention relates to intermediates and their salts useful in thepreparation of the gastrointestinal hormone, secretin. The intermediatesof this invention comprise partial sequences of amino acids which, whencombined, form the secretin molecule. The novel protective peptidescorresponding to these partial sequences are:

(1') Protected L-histidyl-L-seryl-L-aspartglyglycine;

"(amino acids 1 to 4);

(2) Protected L-threonyl-L-phenylalanyl-L-threonyl-L- serine hydrazide;(amino acids 5 to 8);

(3) Protected L-glutamyl-L-leucyl-L-seryl-L-arginyl-L- leucinehydrazide; (amino acids 9 to 13); and

(4) Protected L-arginyl-L-aspartyl-L-seryl-L-alanyl-L-arginyl-L-leucyl-L-glutaminyl-L-arginyl-L-leucyl-L-g1utaminylglycyl-L-leucyl-L-valinamide; (amino acids 1 14 to 27).

This application is a continuation of Ser. No. 73 6,880, filed Apr. 12,1968, which is a division of application Ser. No. 553,290, filed May 27,1966, now Pat. No. 3,400,118.

This invention relates to novel peptides, and more particularly, topeptide intermediates and their salts utilized in the preparation of thegastrointestinal hormone, secretin. Porcine secretin has the formula:

His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu-Glu(NHI)-.Arg-Leu-Leu-Glu-(NHQ-Gly- Leu-Val-NH:

1e 27 and hence it is a peptide containing 27 amino acid residuescontaining the amino acids: L-histidine (His); L- aspartic acid (Asp);L-serine (Ser); glycine (Gly); L- threonine (Thr); L-phenylalanine(Phe); L-glutamic acid (Glu); L-glutamine [Glu(NH L-leucine (Leu); L-

arginine (Arg); L-alanine (Ala); and L-valinamide (Val- NH The abovementioned peptide salts include, for instance, hydrochlorides,hydrobromides, acetates, fluoroacetates, such as trifluoroacetate, andchloroacetates such as dichloroacetate.

This 27 membered chain of amino acids is synthesized by preparingpartial sequences of amino acids and then combining the sequences toform the end product of this invention.

Although the number of amino acids may differ in each sequence, e.g., 2,5, 7, and so forth, in the preparation of secretin, it has been foundthat four sequences of amino acids can satisfactorily combine to givethe desired end product.

3,812,092 Patented May 21, 1974 er lce.

The novel four protected peptides corresponding to these partialsequences are:

(1) protected L-histidyl-L-seryl-L-aspartylglycine;

(amino acids 1 to 4);

(2) protected L-threonyl-L-phenylalanyl-L-threonyl-L- serine hydrazide;(amino acids 5 to 8);

(3) protected L-glutamyl-L-leucyl-L-seryl-L-arginyl-L- leucinehydrazide; (amino acids 9 to 13), and

(4) protected L-arginyl-L-aspartyl-L-seryl-L-alanyl-Larginyl-L-leucyl-L-glutaminyl-L-arginyl-L-leucyl-L-leucyl-L-glutaminylglycyl-L-leucyl-L-valinamide; (amino acids 14 to27).

The above sequences may be joined by any known coupling method ofpeptide synthesis to form polypeptides.- The secretin polypeptide may beobtained by methods known for the synthesis of peptides. It is possibleto join together the amino acids indicated in the above formulae, one ata time or by first forming constituent peptide unit sequences andjoining these together until the hormone secretin results.

Such addition is accomplished by activating the carboxylic acid group inthe amino acid to be added, as by protecting the amino group in suchamino acid by converting it to its benzyloxycarbonyl derivative andconverting it to its nitrophenyl ester derivative, and then interactingthe peptide sequences.

Among the suitable activating groups may be mentioned any group whichcauses the acid function to become more reactive, such as mixedanhydrides (which normally involves the acylation of an amine with themixed anhydrides of, for instance, an acyl amino acid and isovalericacid), azides, acid chlorides, O-acyl hydroxylamine derivatives, andactive esters, such as alkyl esters with electron attracting (negativesubstituents, vinyl esters, enol esters, phenyl ester, thiophenylesters, nitrophenyl esters, 2,4-dinitrophenyl esters, andnitrophenylthiol esters. The use of nitrophenyl esters is particularlypreferred from the standpoint of yield, lack of complicating by-productsand consequent ease of purification.

In forming peptide sequences of this invention, the amino functions maybe protected by commonly used amino protecting groups such asbenzyloxycarbonyl, tertiary butyloxycarbonyl, phthalyl,o-nitrophenylsulfenyl, tosyl, and so forth. Methyl, ethyl, tertiarybutyl, benzyl and so forth may be used to protect the carboxyl groups.The hydroxyl protecting groups may be benzyl, tert. butyl,tetrahydropyranyl and so forth, and the guanidine protecting groups maybe nitro, tosyl, p-nitrobenzyloxycarbonyl, protonation, and so forth.

The protecting groups are removed by known reactions such as reductionwith sodium in liquid ammonia, hydrogenolysis (for instance, in thepresence of a palladium on charcoal catalyst), treatment with ahydrohalo acid (such as hydrobromic or hydrochloric acids) in aceticacid or treatment with trifluoroacetic acid.

To prepare the free amines after treatment with a hydrohalo acid inacetic acid, the hydrobromide salt is treated either with an ionexchange resin such as Amberlite IR400 or so neutralized with an aminesuch as triethylamine.

Specific compounds of the present invention are compounds of the formulaand physiologically acceptable acid addition salts thereof and carboxylactivated derivatives thereof wherein R is hydrogen, an N-terminal aminoprotecting group or N- terminal protected L-histidyl-L-seryl wherein theN-terminal protecting groups are benzyloxycarbonyl, t-butyloxycarbonyl,phthalyl, o-nitrosulfenyl or tosyl, and R is a 13- carboxyl protectinggroup selected from the group consisting of lower alkyl or benzyl,provided that when R is benzyloxycarbonyl, R is not methyl.

EXAMPLE 1 Benzyloxycarbonyl-L-valinamide (I) Benzyloxycarbonyl-L-valinep-nitrophenylester (M.P. 67-68", 18.6 g.) is suspended in methanol (50ml.) to dissolve part of the material. A methanolic solution of ammonia(40 ml., ca. 3 N) is added. A clear yellow solution results andcrystallization of the product starts after a few minutes. After onehour at room temperature, the crystals are filtered and washed withmethanol (ca. 50 ml.) and with ethyl acetate (ca. 50 ml.). The air driedproduct (8.25 g.) melts at 206-208". From the mother liquor, morematerial (0.95 g.) is secured, M.P. 206- 207. Total yield, 9.2 g.(73.5%). A sample (3.5 g.) is recrystallized from ethyl acetate (450ml.). The purified amide (3.0 g.) melts at 206-208, [u] .+22 (c. 2dimethylformamide). The melting point is unchanged after sublimation at190 and 0.05 mm. Hg.

Analysis.Calcd. for I, C H N O C, 62.4; H, 7.2; N, 11.2; Found: C, 62.4;H, 7.3; N, 11.2.

EXAMPLE 2 Benzyloxycarbonyl-L-leucyl-L-valinamide (II)Benzyloxycarbonyl-L-valinamide (I) (12.5 g.) is suspended in acetic acid(50 ml.) and treated with hydrobromic acid in acetic acid (ca. 4 N, 50ml.). Soon a clear solution is obtained and evolution of gas (CO can beobserved and later also the deposition of crystals. After one and a halfhours at room temperature, ether (250 ml.) is added and the crystallinehydrobromide is filtered off, washed with ether (125 ml.) and dried oversodium hydroxide in. vacuo. The yield is quantitative (9.8 g.), M.P.248-253. The amide hydrobromide is dissolved in dimethylformamide (100ml.), triethylamine ml.) is added to the solution followed bybenzyloxycarbonyl-L- leucine p-nitro-phenylester (23.2 g.). Some heatevolution will be observed. The mixture is cooled to room temperature;after a few minutes, it shows no ninhydrin reaction and soon it turnsinto a semi-solid mass of crystals. After three hours at roomtemperature, the mixture is diluted with ethyl acetate (300 ml.),filtered and the crystals washed with ethyl acetate (200 ml.),chloroform (300 ml.) and again with ethyl acetate (200 ml.). The airdried product (15.8 g.) sinters at 225 and melts at 232-234". From themother liquors a small second crop (0.25 g.) is obtained, M.P 232-233.Total yield, 88%. Recrystallization of a sample from hot 95% ethanolraises the M.P. to 234-236", 1 (c. 2.5 dimethylformamide). The protecteddipeptide amide sublimes unchanged at 220 and 0.5 mm. Hg.

Analysis.-Calcd. for II, C H N O C, 62.8; H, 8.0; N, 11.6. Found: C,62.9; H, 8.1; N, 11.6.

EXAMPLE 3 Benzyloxycarbonyl-glycyl-L-leucyl-L-valinamide (III) To asuspension of the protected dipeptide amide (II), (14.6 g.) in aceticacid (40 ml.) hydrobromic acid in acetic acid (ca. 4 N, 40 ml.) isadded. After one and a half hours at room temperature, the mixture isdiluted with ether (400 ml.). A semisolid mass forms and is treated withfresh ether until it disintegrates. The hydrobromide is filtered off,washed with ether and dried in vacuo over sodium hydroxide for one hour.It is dissolved in dimethylformamide 100 ml.), triethylamine (14 ml.)and benzyloxycarbonylglycine p-nitrophenylester (16.5 g.) are added tothe solution. Some evolution of heat is observed and the mixture iscooled to room temperature where it is kept for about three hours. Theprecipitate (triethylammonium bromide) is filtered off and washed withdimethylformamide (50 ml.). Ethyl acetate (500 ml.) and N HCl (400 ml.)are added to the filtrate and the organic layer is washed with N HCl(400 ml.) and with water (twice 400 ml.). The aqueous washes areextracted in the same order with a portion (ca. 300 ml.) of "ethyl'acetate. From the combined ethyl acetate solutions, a precipitate slowlyforms. Most of the solvent is removed by evaporation and the protectedtripeptide amide is washed with ethyl acetate. The dry product weighs16.8 g. (quantitative yield), M.P. 170-175 with some sintering at 160.This crude product is extracted with boiling ethyl,

' EXAMPLE 4 Benzyloxycarbonyl-L-glutaminylglycyl-L-leucyl-L- valinamide(IV) The benzyloxycarbonyl group is removed fromthe protected tripeptideamide (III) (12.7 g.) as described in the previous paragraph. To thesolution of the hydrobromide in dimethylformamide ml.), triethylamine(11.2 ml.)

and benzyloxycarbonyl-L-glutamine p-nitrophenylester (13.25 g.) areadded. The mixture, which is cooledwith water to keep it at roomtemperature, soon turns into a semisolid mass of crystals. Afterstanding overnight at room temperature, the mass is disintegrated withthe aid of chloroform (600 ml.). The product is filtered and washed onthe filter with chloroform (200 ml.), with warm ethyl acetate (200 ml.),warm ethanol (200 ml.) and again with warm ethyl acetate (300 ml.). Theprotected tetrapeptide amide is dried at 50 in vacuo; Weight is 16.0 g.yield), M.P. 239-241 dec., [(11 -13 (c. 2 dimethylformamide).

Analysis.-Calcd for IV, C H N O C, 5.6.9; H, 7.3; N, 15.3. Found: C,56.9; H, 7.3; N, 15.5.

EXAMPLE 5 Benzyloxycarbonyl-L-leucyl-L-glutaminylglycyl-L-lucylQL-valinamide (V) The protected tetrapeptide amide (IV) (13.8 g.)issuspended in acetic acid (40 ml.) and treated with hydrobromic acid inacetic acid (ca. 4 N, 40 ml.). After two hours at room temperature, thereaction mixture is diluted with ether (650 ml.), the hydrbromide isfiltered and washed with ether (400 ml.). After short drying in vacuoover so dium hydroxide, the hydrobromide is dissolved indimethylformamide (75 ml.) and triethylamine (2.5 ml.) is add:

ed to the solution followed by benzyloxycarbonyl-L-leucine mother liquorand washings, M.P. 256-259 dec. This is recrystallized from aceticacid-95% ethanol to give a prod uct (0.50 g.) with M.P. 263-270 dec.Total yield is 14.0 g. (84%).

Analysis.-Ca1cd. for V, C H N O C: 58.1, H: 7.8, N: 14.8. Found: C:58.1, H: 7.8, N: 14.6.

EXAMPLE 6 L-leucyl-L-glutaminylglycyl-L-leucyl-L-valinamide hydrobromide(Va) 7 Compound V (3.3 g.) is dissolved in hot acetic acid (20 ml.), thesolution is cooled to room temperature and bydrobromic acid in aceticacid (ca. 4 N, 10 ml.) added.

After one hour at room temperature, the solution is diluted with ether(250 ml.), the gummy precipitate disintegrated under fresh ether andwashed with ether on the filter. The crude hydrobromide is :dried in adesiccator over sodium hydroxide overnight. It is dissolved in methanol(50 ml.) and precipitated with ether (150 ml;). The gummy precipitate isdissolved in absolute ethanol.(ca. 200 ml.). A crystalline precipitateseparates slowly ,from

the solution. Triethylamine (ca. 1 ml.) is added to neutralize thesolution. The crystals are washed with ethanol (100 ml.) and dried overP in vacuo at room temperature. Weight is 0.50 g., M.P. 2152l6 dec.after sintering at 210. Evaporation of the mother liquor to dryness andtreating of the residue with chloroform (50 ml.) gives a second crop,1.75 g., M.P. which is similar to the first crop. A small third crop(0.35 g.) is obtained from the mother liquors of the second, M.P.220-222" dec. On paper chromatograms in the system of butanol: aceticacid: water (4: 1:5), a single spot (R =0.60)-is shown by all thefractions. Total yield is 2.60 g. (86% A sample (0.50 g.) is dissolvedin hot 95% ethanol (25 ml.). On standing, slow crystallization occurs; afew days later, ethyl acetate (25 ml.) is added and two days later thecrystals are filtered oif, washed with a 1:1 mixture of ethanolethylacetate (20 ml.) and with ethyl acetate (20 ml.). After drying, thecrystals weigh 0.40 g., M.P. 220 dec. (sinters at 200). A sample isdried at 110 for analysis.

Analysis.--Calcd. for VI, C H N O Br: C: 47.2, H: 7.7, N: 16.1, Br:13.2. Found: C: 47.2, H: 7.7, N: 15.9 Br: 13.0.

Amino acid analysis:

EXAMPLE 7 Glu, 0.9; Gly, 1.1; 1.0; Leu,

L-leucyl-L-glutaminylglycyl-Ldeucyl-valinarnide (Vb) The pentapeptideamide hydrobromide (Va) (0.30 g.) is dissolved in methanol (50 ml.) andtreated with Amberlite IR400 in OH cycle until the solution gives anegative test with silver nitrate. The resin is removed by filtration,washed with methanol and the solvent removed in vacuo. The residue istreated with chloroform (5 ml.) in which it partially dissolves. Ethylacetate (50 ml.) is added. The product is filtered and washed with ethylacetate (25 ml.) and dried in air, weight is 0.20 g., M.P. 260- 261 dec.The product gives a single spot, R =0.60 on paper chromatograms.

Analysis.Calcd. for VII, C H N O C: 54.6, H: 8.6, N: 18.6. Found: C:55.0, H: 8.6, N: 18.6.

EXAMPLE 8Benzyloxycarbonyl-L-leucyl-L-leucyl-L-glutaminylglycyl-L-leucyl-L-valinamide7 (VII) The pentapeptide amide hydrobromide (Va) (1.22 g.) is suspendedin dirnethylformamide ml.) in which it partially dissolves.Benzyloxycarbonyl-L-leucine p-nitrophenylester (1.55 g.) is added to themixture which is shaken vigorously for a few minutes, diluted withdimethylformamide (10 ml.), and shaken again. After two hours at roomtemperature, the mixture is diluted with ethyl acetate (150 ml.),filtered and washed with ethyl acetate (20 ml.). The precipitateissuspended in absolute ethanol (30 ml.),washed on the filter withabsolute ethanol (20 ml.) and with ethyl acetate (20 ml.). After drying,the protected hexapeptide amide weighs 1.55 g. (100%); the productsinters at 255, melts with decomposition at 25 8-262.

Analysis.-Calcd. for VI, C H N O' C: 58.9, H: 8.1, N: 14.5. Found: C:58.3, H: 7.9, N: 13.9.

EXAMPLE 9 L-leucyl-L-leucyl-L-glntaminylglycyl-L-leucyl-L-valinamidehydrobromide (VIa) The protected hexapeptide amide (VI), (0.39 g.) is

suspended in acetic acid (5 ml.) and treated with hydrobromic acid inacetic acid (4 N, 5 ml.). Soon a clear solution results. After one and ahalf hours at room temperature, ether (200 m1.) is added; theprecipitate is filtered, washed with ether and dried in vacuo oversodium hydroxide. The salt is hygroscopic. Since it gives a single spot(R =0..75) with ninhydrin on paper chromatograms in butanol-aceticacid-water (4: l :5), it is used without purification.

EXAMPLE 10 L-leucyl-L-leucyl-L-glntaminylglycyl-L-leucyl-L- valinamide(VIb) The hexapeptide amide hydrobromide (VIa) (from 0.39 g. protectedpeptide) is converted to the free base as-described in Example 7. Theproduct (0.29 g.) separates in crystalline form. On paper chromatograms,it shows a single spot with R;=0.75, M.P. 239-241 dec.

Analysis.Calcd. for X, G l-1 N 0 C: 56.2, H: 8.8, N: 17.5. Found: C:56.1, H: 8.9, N: 16.9.

A sample is hydrolyzed with constant boiling hydrochloric acid in anevacuated, sealed ampoule at for 24 hours; it gives 5.1% NH calcd. 5.3%.Quantitative aminoacid analysis gives the following molar ratios: Leu,3.0; Glu, 1.1;Gly,1.1;Val,1.1.

EXAMPLE 11 Benzyloxycarbonyl-nitro-L-arginyl-L-leucyl-L-leucyl-L-glutaminyl-glycyl-L-leucyl-L-valinamide (VII) A solution of N-benzyloxycarbonyl-nitro-L-arginine (3.53 g.) and of 2,4-dinitrophenol(2.0 g.) in'tetrahydrofuran (60 ml.) is cooled with ice water during theaddition of dicyclohexylcarbodiimide (2.1 g.). After about one hour atroom temperature, the precipitate (dicyclohexylurea) is filtered off andis washed with tetrahydrofuran (40 ml.). The combined filtrate andwashings are evaporated to dryness in vacuo, the residue dissolved inethyl acetate (ca. 10 ml.) and precipitated with ether (ca. 50 ml.). Theester is washed with ether (ca. 50 ml.) and dried in vacuo.

The hexapeptide amide (VIb) (from 3.9 g. protected hexapeptide amide bythe procedure of Example 10 is dissolved in hot (ca. 80)dimethylformamide ml.) and is mixed with a solution of the above activeester in the same solvent (50 ml.); After about four hours at roomtemperature, the mixture is diluted with ether (1 liter), theprecipitate filtered, washed with ether (0.6 liter), ethyl acetate (0.3liter) and dried. The product, weight 4.5 g. (92%), M.P. 240-242 (dec.),-38 (c. 2 acetic acid), is shown to be homogeneousby treatment of asmall sample with hydrobromic acid in acetic acid, precipitation of thehydrobromide with ether and paper chromatography of this material. Asingle spot, R =0.83, is observed in the n-butanol-acetic acid-water(4:1:5) system.

A sample, 0.20 g., is recrystallized from hot 50% ethanol (22 ml.), thecrystals are washed with ethanol and ethyl acetate. 0.12 g. isrecovered, M.P. ca. 252 dec.,

A 269 my, E12,, 144

Analysis.-Calcd. for C H N O C: 54.2,1-1: 7.5, N: 18.7. Found: 54.3, H:7.6, N: 18.3.

EXAMPLE 12 Benzyloxycarbonyl L glutaminyl-nitro-L arginyl L- leucyl Lleucyl L glutaminylglycyl L leucyl-L- valinamide (VIII) Y at roomtemperature for about three hours. The mixture is diluted with ethylacetate (1 liter), the precipitate is filtered and Washed with ethylacetate (0.5 liter), ethanol (0.5 liter) and with ethyl acetate, hotethyl acetate (0.25 liter) and hot chloroform (0.25 liter).-'Ihe productis dried first in air and then at 50 in vacuo, weight 10.3 g. (93%),darkens from 250 dec. at 262-264. A sample (1.0 g.) is dissolved in hot80% ethanol ml.), cooled, and diluted with 95% ethanol ml.). Slowcrystallization took place. After three days the crystals are filtered,washed with ethanol, chloroform and ethyl acetate. The dried materialweighs 0.85 g., M.P. 250 dec., 1] -32 (c. 2 dimethylsulfoxide).

Analysis.Calcd. for C4QH81N15O14: C: 53.3, H: 7.4, N: 19.0. Found: C:53.4, H: 7.5, N: 19.2.

EXAMPLE 13 Benzyloxycarbonyl L leucyl L glutaminyl nitro-L- arginyl Lleucyl L leucyl L glutaminylglycyl- L-leucyl-L-valinamide (IX) Theprotected octapeptide (VIII) (11.1 g.) is powdered and added withstirring to acetic acid ml.). Hydrobromic acid in acetic acid (ca. 4 N,50 ml.) is added to the suspension, and stirring is continued until allthe material dissolved and then for 30 minutes more. A total of threehours are required. The amine hydrobromide is precipitated with ether(1.3 liters), filtered, washed with ether and dried in vacuo over sodiumhydroxide for a short time. The hydrobromide is dissolved in'dimethylformamide 100 ml.), and triethylamine (10.4 ml.) is added tothe cooled solution, followed by the benzyloxycarbonyl- L-leucinep-nitrophenylester (5.0 g.). After about three hours at room temperaturethe mixture is diluted with ethyl acetate (2 liters). The precipitatewhich formed is filtered and washed with 200 ml. portions of ethylacetate, chloroform, hot chloroform and hot ethyl acetate. The productis dried in vacuo at 50'; weight 11.15 g. (91.5%) MP. darkens from 255dec. at about 265. In a second similar preparation, a quantitative yieldis obtained. Treatment of a sample (50 mg.) of (IX) with hydrobromicacid in acetic acid, precipitation of the free nona-peptide amidehydrobromide with ether, and paper chromatographic examination of theproduct in the system butanolacetic acid-water, 4:1:5, reveals a singlespot (R;=0.77).

A sample of (IX) (0.50 g.) is dissolved in a hot mixture of ethanol (30ml.) and water (15 ml.). The solution is filtered and allowed to standat room temperature. After a few days, the crystals are collected,washed with liberal quantities of 95% ethanol, ethyl acetate and hotethyl acetate. The air dry material (025 g.) darkens from 260, meltswith dec. at 267-269". For analysis, it is dried at 110 in vacuo.

Analysis.--Calcd for CH92N16O15: C: 54.3, H: 7.6, N: 18.4. Found: C:54.2, H: 7.8,N: 18.2.

EXAMPLE l4 Benzyloxycarbonyl nitro L arginyl L leucyl L-glutaminyl-nitro L arginyl L leucyl L leucyl-L-glutaminylglycyl-L-leucyl-L-valinamide (X) The protected nonapeptideamide (IX) (9.8 g.) is suspended in acetic acid (40 ml.) and hydrobromicacid in acetic acid (ca. 4 N, 40 ml.) is added to the suspension. Ahomogeneous solution is obtained in about one hour, and the solution iskept at room temperature for an additional hour. Ether (900 ml.) isadded to the solution. The precipitated hydrobromide is filtered, washedwith ether and dried in vacuo over sodium hydroxide for a short time. Itis dissolved in dimethylformamide (80 ml.); the solution is cooled whilebeing made alkaline with triethylamine (5.5 ml.). Benzyloxycarbonylnitro-L- arginine 2,4-dinotrophenyl ester (10.3 g.) (prepared asdescribed in the preparation of VII) is added followed by moretriethylamine (2.2 ml.). After three hours at room temperature when thesolution gives no' reaction with ninhydrin, it is diluted with ethylacetate (1 liter). Next day the precipitate is collected on a filter andis washed with ethyl acetate (200 ml.), chloroform (200 ml.), hotchloroform (750 ml.), abs. ethanol (200 ml.), and ethyl acetate (200ml.). The product (8.2 g., 72%) darkens from 250 and melts with dec. at255-264". The benzyloxycarbonyl group is removed from a small samplewith hydrobromic acid in acetic acid; the resulting free amine gives asingle spot on paper chromatograms with R;=0.80 in the system ofbutanol-acetic acid-water (4:1: 5) or with R =0.60 in the system ofbutanol pyridineacetic acid-water (30:20:6z24).

Analysis.Cald. for CuHmaOmNn (M.W. C: 51.7, H: 7.3, N: 20.7. Found: C:51.9, H: 7.6, N: 20.1.

EXAMPLE 15 Benzyloxycarbonyl L alanyl-nitro-L-arginyl-L-leucyl- Lglutaminyl nitro L-arginyl-L-leucyl-L-leucyl-L-glutaminylglycyl-Izleucyl-L-valinamide (XI) The protected decapeptideamide (X) (5.68 g.) is dissolved in acetic acid (30 ml.) and treatedwith a solution of hydrobromic acid in acetic acid (ca. 4 N, 30 ml.).After one and one-half hours at room temperature, the decapeptidehydrobromide is precipitated with ether (600 ml.), washed with ether anddried briefly in vacuo over sodium hydroxide pellets. The hydrobromideis dissolved in dimethylformamide (40 ml.); triethylamine (4.6 ml.) isadded to the solution followed by benzyloxycarbonyl-Lalaninep-nitrophenylester (1.72 g.). Finally the mixture is made alkaline withmore triethylamine (0.5 ml.). The reaction is allowed to proceedovernight, then the mixture is diluted with chloroform. The addition of250 ml. of this solvent results in a clear solution, with a second 250ml. portion a crystalline precipitate forms. This is collected, washedwith chloroform (200 ml.) and ethyl acetate ml.). The dry product (4.85g., 81.5%) melts at 258-264" dec.

Analysis.Calc'd. for C H N O C: 51.6, H: 7.3, N: 20.7. Found: C: 51.0,H: 7.5, N: 19.9.

EXAMPLE 16 L Aspartyl L seryl L-alanyl-L-arginyl-L-leucylleucyl Lglutaminyl L -arginyl-L-leucyl-L-leucyl-L-gI-utaminylglycyl-L-leucyl-L-valinamide (XIIIa) (l) L Alanyl Larginyl-L-leucyl-L-glutaminyl-L- arginyl L leucyl L leucylL-glutaminylglycyl-L- leucyl-L-valinamide (acetate) (XIa): The protectedhendecapeptide (XI) (3.0 g.) is dissolved in 86% acetic acid (100 ml.)and hydrogenated in the presence of a 10% palladium on charcoal catalyst(1.5 g.) for two days. The catalyst is removed by filtration and thesolvents by evaporation in vacuo. A sample of this free hendecapeptideacetate (XIa) is examined on paper chromatograms in the system ofn-butanol-acetic acidwater (4:1:5), where it gives a single spot(R,0.15) both with ninhydrin and the Sakaguchi reagent. Similarly, asingle spot (R -0.6) is revealed in the system nbutanolaceticacid-pyridine-water 30 6 20:24)

(2) Benzyloxycarbonyl L seryl L-alanyl-L-arginyL L leucyl Lglutaminyl-L-arginyl-L-leucyl-L-leucyl-L-g1utaminylglycyl-L-leucyl-L-valinamide (acetate) (XII): To the freehendecapeptide (XIa) is dimethylformamide (40 ml.)benzyloxycarbonyl-L-serine 2,4-dinitrophenyl ester (1.0 g.) is added.The mixture, a gel, is filtered through a glass filter, and undissolvedmaterial on the filter is brought into solution with small portions ofhot dimethylformamide. A total of 60 ml. of this solvent is used. Afterthree hours, more of the active ester (0.6 g.) is added. After anadditional hour, the mixture gives no reaction with ninhydrin. Next daythe mixture is diluted with ethyl acetate (900 ml.), the resulting oilyprecipitate is triturated with ethyl acetate (1 liter), the solidifiedprotected dodecapeptide (XII) is filtered and washed with ethyl acetate.The product (2.1 g.) is impure as shown by its broad M.P.: sinter at 73,melts with dec. at 163. Evaporation of the mother liquor and triturationof the residue with ethyl acetate gives an additional quality (0.97 g.)of material with a similar melting point.

(3) The crude protected dodecapeptide (XII) (0.65 g.) is dissolved in amixture of water (40 ml.) and acetic acid (4 ml.) and the solution isextracted with ethyl acetate (three 20 ml. portions). The ethyl acetateextracts are washed with water ml.). The aqueous wash is pooled with themain aqueous solution; a 10% palladium on charcoal catalyst (0.2 g.) isadded and the mixture is hydrogenated for about four hours. After theremoval of the catalyst by filtration, the solution is lyophilized. Thecrude free dodecapeptide Weighs 0.46 g. A similar preparation (from 2moles of protected dodecapeptide) is dissolved in dimethylformamide (10ml.) and treated with benzyloxycarbonyl-L-aspartic acid fi-benzyl,11,1)- nitrophenylester (1.44 g.). Next day a second portion (0.48 g.)of the active ester is added to the mixture and after three more hours,a last portion (0.48 g.). After three more hours at room temperature,ethyl acetate (400 ml.) is added; the precipitate which formed isfiltered and washed with ethyl acetate (200 ml.). The material, whichturns into a crystalline mass on the filter, weighs 2.65 g., M.P.200-220 dec. This crude preparation (1.3 g.) is dissolved in a mixtureof acetic acid (40 ml.) and water (40 ml.), a 10% palladium on charcoalcatalyst (0.65 g.) is added and the mixture is hydrogenated overnight.After removal of the solvent by filtration and the solvents byevaporation in vacuo, the residue is distributed in a system ofn-butan0l0.2% dichloroacetic acid in water, through 60 transfers. Fortyml. lower and upper phases are used. The material in tubes No. 16-26 ispractically pure as shown on paper chromatograms, which reveals thepresence of a trace of aspartic acid in these fractions. Tubes No. 0-7contain mostly aspartic acid. In tubes 8- 15, the free tridecapeptidecontaminated with more aspartic acid is present. A fast moving,ninhydrin-negative, Sakaguchi-positive impurity travels close to thefront and is easily separated from the desired material. The contents oftubes No. 16-26 are pooled, the solvents evaporate to a small volume invacuo, and the free tridecapeptide (XIIIa) is isolated by lyophilization(170 mg). After hydrolysis with constant boiling hydrochloric acid at110 for 16 hours in vacuo, a sample shows the following ratios of aminoacids: Glu, 2.0; Asp ,1.4; Ser, 0.9; Gly, 1.1; Ala, 1.1; Val, 1.0; Leu,3.8; NH 2.9; Arg, 1.9. Examination of a sample by paper electrophoresis(collidine acetate, pH 6.3, and triethanolamine acetate, pH 4.0) revealsa small amount of aspartic acid as an impurity, but the peptide travelsas a single component towards the cathode.

The fast moving, ninhydrin-negative, Sakaguchi-positive impurity, whichis separated in the above mentioned counter-current distribution, onamino acid analysis reveals that it contains only the amino acidspresent in the hendecapeptide derivative (XI). Calc'd. for the acetylderivative of the free hendecapeptide (di-dichloroacetate): N: 17.8, CI:9.1, CH CO: 8.2. Found: N: 17.1, C1: 9.5, CH CO: 7.0.

(l) Tert. butoxylcarbonyl L aspartic acid a-p-nitrophenyl, fl-benzylester: To a cooled solution of tert.- butoxycarbonyl-L-aspartic acidfi-benzyl ester (16.2 g.) and of p-nitrophenol (8.5 g.) in ethyl acetate(150 ml.) dicyclohexylcarbodiimide (10.3 g.) is added. After two hoursat room temperature, acetic acid (0.5 ml.) is added, thedicyclohexylurea is removed by filtration and washed with water and thecombined filtrates are evaporated in vacuo. The oily residuecrystallizes on the addition of 95% ethanol. The crystals are collected,washed with ethanol and dried. Evaporation of the mother liquor givesmore of the same active ester. A total of g. is obtained, M.P.

10 -81 [011 36 (c. 2 dimethylformamide containing 1% acetic acid).

Analysis.-Calcd. for C H O N N: 6.30. Found: N: 6.34.

(2) N tert. butyloxycarbonyl O-benzyl-L-seryl-L- alanylnitro L arginyl Lleucyl-L-glutaminyl-nitro-L- arginyl L leucyl L leucyl Lglutaminylglycyl-L- leucyl L valinamide (XIIb): To a suspension of theprotected hendecapeptide (XI) (6.4 g.) in acetic acid (45 ml.), a 4 Nsolution of hydrobromic acid in acetic acid (45 ml.) is added. Afterabout 2 hours at room temperature, the solution is diluted with ether(650 ml.). The

gummy precipitate is triturated with ether, filtered, washed with etherand dried in vacuo over sodium hydroxide. The hydrobromide is dissolvedin methanol (40 ml.) and reprecipitated with ether (300 ml.). Thesemisolid mass is triturated with ether, filtered, washed with ether anddried as before. The solid material is suspended in methanol (200 ml.)and enough triethylamine (5 ml.) is added to render the mixture slightlyalkaline. The solvent is removed in vacuo and the residue extracted withchloroform (50 ml.). The insoluble material is dried in vacuo: 6.1 g.,M.P. 242-248" dec. In a system of n-butanol-acetic acid-water (4:1:5), asingle spot (R =0.62) is revealed with ninhydrin and by U.V. absorption.In the system butanol-pyridine-acetic acid-water (30:20:6:24) a singlespot is also found (R,=0.72). The hydrobromide is partially'dissolved indimethylformamide (20 ml.), triethylamine (0.6 ml.) and N tert.butyloxycarbonyl-O-benzyl- L-serine p-nitrophenyl-ester indimethylformamide (20 ml.) is added. The active ester is prepared fromthe corresponding acid (2.55 g.), p-nitrophenol (1.5 g.) anddicyclohexylcarbodiimide (1.7 g.) in ethyl acetate (21 ml.). The oilwhich is left after the removal of the dicyclohexylurea by filtrationand of the solvent by evaporation is used without purification. Themixture is filtered and the yet undissolved material is dissolved withhot dimethylformamide (100 ml.). The mixture is allowed to standovernight at room temperature. The protected dodecapeptide is isolatedby the addition of ethyl acetate 1.5 liters); the precipitate isfiltered and washed with ethyl acetate (0.5 liter). The crude product(5.14 g.) after darkening at 255 melts with dec. at 298.

A sample (1.0 g.) of this material is suspended in ethanol (25 ml.),heated to boiling for a few minutes, and allowed to cool. During thistreatment the solid material changes into a crystalline mass. Thepurified material is washed with 95% ethanol and dried. The product(0.78 g.) darkens from 255 and melts with dec. at 298-304.

(3) Tert.-butyloxycarbonyl B benzyl L aspartyl- O-benzyl L seryl Lalanyl nitro L arginyl L- leucyl L glutaminyl-nitro L arginyl -Lleucyl-L- leucyl L glutaminylglycyl- L leucyl L valinamide (XIII): Theprotected dodecapeptide amide (XII) (3.3 g.) is dissolved intrifiuoroacetic acid (32 ml.). After about fifteen minutes most of thetrifiuoroacetic acid is evaporated in vacuo and the syrupy residuediluted with ether (300 ml.). The precipitate is collected, washed withether and dried in vacuo over sodium hydroxide. The free amine,trifiuoroacetate (3.2 g.), has no well defined melting point; it softensat about 135, darkens from 240 and decomposes at 304. On paperchromatograms in the system butanol-acetic acid-water (4:1:5), a singlecomponent (R =0.52) is revealed by U.V. absorption and by ninhydrin.This material is dissolved in dimethylformamide (250 ml.), triethylamine(0.28 ml.) and tert.- butyloxycarbonyl ,3 benzyl L aspartic acidp-nitrophenylester (1.76 g.). The solution is concentrated in vacuo toabout ml. Next day more triethylamine (0.28 ml.) andtert.-butyloxycarbonyl 3 benzyl L aspartic acid p-nitrophenylester (0.88g.) are added. One day later the mixture gave no more reaction withninhydrin. It is diluted with ethyl acetate (500 ml.) the precipitate isfiltered, washed with ethyl acetate and dried at room temperature. Theprotected tridecapeptide softens and 11 darkens from about 250 and meltswith dec. at 307- Analysis.-Calcd. for C82H132N24O24: CI 53.6, H: 7.6,N: 18.3. Found: C: 54.3, H: 7.2, N: 18.5.

(4) A sample of the fully protected tridecapeptide amide (XXII) (40 mg.)is dissolved in acetic acid (4 ml.); a 10% palladium on charcoalcatalyst (40 mg.) is added and the mixture hydrogenated for 2 days. Thecatalyst is filtered oil and the acetic acid removed by evaporation fromthe frozen state. The residue is dissolved in acetic acid (1 ml.) and asolution of hydrogen chloride in acetic acid (2.6 N, 0.5 ml.) is added.Soon an oily precipitate forms. After about one-half hour the mixture isdiluted with ether and the precipitate, which solidifies, is washed withether. The white solid material (hydrochloride) is shown to behomogeneous on paper chromatograms in the systems of butanol-aceticacid-water (421:5) and butanol-acetic acid-pyridine-water (30:6:20224).In this system the tridecapeptide travels as a single spot with R,values 0.32 and 0.28 respectively. The spots are revealed with ninhydrinand with the Sakaguchi reagent. On paper electrophoresis incollidineacetate (pH 7) the material travels as a single band to wardthe cathode and is indistinguishable from the product obtained byprocedure (a).

EXAMPLE 17 N-benzyloxycarbonyl 8 benzyl L aspartyl-O-benzyl- L-seryl Lalanyl-nitro L arginyl L leucyl-L- glutaminyl-nitro L arginyl L leucyl Lleucyl- L-glutaminylglycyl-L-leucyl-L-valinamide (XIIIb) The protecteddodecapeptide amide (XIIb) (0.82 g.) is suspended in acetic acid (24ml.). A solution of hydrochloric acid in acetic acid (2.6 N, 15 ml.) isadded. This causes the dodecapeptide to dissolve, but soon thecorresponding amine hydrochloride deposits as an oil. After one-halfhour at room temperature, ether (ca. 200 ml.) is added to the mixture;the residue, which solidifies is washed with ether and dried over sodiumhydroxide in vacuo. This hydrochloride is suspended in dimethylformamide(ca. 10 ml.) and treated with triethylamine (0.2 m1.) andN-tert.-butoxycarbonyl L aspartic acid 13- benzyl-,a-p-nitro-phenylester (0.44 g.). The mixture is filtered from insolublematerial, the filter being rinsed with dimethylformamide (5 ml.). Afterabout 16 hours at room temperature, the mixture is diluted with ether(ca. 200 ml.). The protected tridecapeptide is washed with ether anddried; 0.66 g. of a product, M.P. 250-255 are obtained. Catalyticreduction of (XIIIb) followed b treatment with hydrochloric acid inacetic acid yields the free tridecapeptide (XIIIa).

EXAMPLE 18 N"-tert.-butyloxycarbonyl-nitro-L-arginyl ,8 benzyl-L-aspartyl-O-benzyl-L-seryl-L-alanyl-nitro L arginyl-L-leucyl-L-glutaminyl-nitro-L-arginyl L leucyl-L-leucyl-L-glutaminylglycyl L leucyl L valinamide The protectedtridecapeptide (XIII) (0.92 g.) is dissolved in trifluoroacetic acid (18ml.). After about minutes at room temperature, most of thetrifiuoroacetic acid is removed in vacuo and the residue is trituratedwith ether (100 ml.) The free amine-trifluoroacetate is filtered, washedwith ether and dried in vacuo over sodium hydroxide. Paperchromatographic examination of this material in the system ofbutanol-acetic acid-water (4:1:5) reveals a single (ninhydrin yellow)component at R =0.78.

To a solution of the trifiuoroacetate in dimethylformamide (15 ml.),tert.-butyloxycarbonyl-nitro-L-arginine 2,4-dinitrophenylester (0.43 g.)(prepared by the method of Example 11, substitutingt-butyloxycarbonyl-nitro-L- arginine for thebenzyloxycarbonyl-nitro-L-arginine) is added followed by triethylamine(0.07 ml.). The mixture is concentrated in vacuo to about 6-8 ml. and asecond portion of triethylamine (0.07 ml.) is added. The next day themixture is further concentrated in. vacuo, and the residue is trituratedwith ethyl acetate. The protected tetradecapeptide is collected on afilter and is washed with ethyl acetate and with ether. The crudeproduct (0.96 g.) sinters at about darkens from 220 and decomposes at305. An aliquot of this material (0.75 g.) is dissolved in the twolayers (each 30 ml.) of the solvent system n-butanol-pyridine-aceticacid-water (4:2:1z7). The solution is placed in the first three tubes ofa Craig apparatus and countercurrent distribution is carried out throughone hundred transfers. Essentially all the material is found in a bandcorresponding to a distribution coeflicient of 11.5, and theexperimental curve is found to be practically identical with the curvecalculated for this K value. The recovered material shows the same M.P.as the one before distribution.

AnalySis.Calcd. for C H N O C: 51.8, H: 7.1, N: 19.9. Found: C: 52.2, H:6.7, N: 19.8.

The protected tetradecapeptide (XIV) (8.2 g.) is dissolved intrifluoroacetic acid (4 ml.). After 15 minutes at room temperature, thesolution is concentrated in vacuo to a syrup, ether (500 ml.) is addedand the precipitate is Washed on a filter with ether. The air driedtrifluoroacetate (XIVa), (8.1 g.) softens at 140 and melts (dec.) at250. In butanolacetic acid-water (4:1:5), a single spot, Rg=0.70 isobserved.

Analysis.-Calcd.: N: 19.8, F: 2.8. Found: N: 20.4, F: 3.3.

EXAMPLE 19 N-L-leucyl-N'-benzyloxycarbonyl hydrazine, trifluoroacetate(XV) Tert.-butyloxycarbonyl-L-leucine monohydrate (10 g.- 40 mmoles) andbenzyloxycarbonyl hydrazine (6.64 g.- 40 mmoles) are dissolved indichloromethane (100 ml.). The solution is cooled in an ice-water bathand added with dicyclohexylcarbodiimide (8.25 g.-40 mmoles). After onehour stirring in the cold bath and five hours at room temperature, theprecipitate of dicyclohexylurea is filtered off and washed with ethylacetate. The filtrate is concentrated to dryness in vacuo, the residueis dissolved in ethyl acetate ml.) and the solution is washed once with20% aqueous citric acid, once with water, twice with saturated sodiumbicarbonate and three times with water. The organic phase is dried overmagnesium sulfate and concentrated to dryness in vacuo. The solidresidue is dissolved in cold trifiuoroacetic acid (35 ml.) and thesolution is kept at room temperature for 15 minutes. After removing mostof the trifiuoroacetic acid in vacuo at room temperature, the oily.residue is dissolved in ether (ca. 40 ml.) and the crystallinetrifluoroacetate is precipitated by addition of hexane (ca. 1 liter).Yield 13.2 g. (84%), M.P. (180) 182-183.

Analysis.-Calcd. for C ,I-I N O -CF COOH: C: 49.0, H: 5.65, N: 10.7, F:14.5. Found: C: 48.4, H: 5.52, N: 10.6, F: 14.3.

EXAMPLE 20 N- (tert.-butyloxycarbonylnitro-L-arginyl-L-leucyl -N'-benzyloxycarbonyl hydrazine (XVI) N-(L-leucyl-N'-benzyloxycarbonylhydrazine trifluoroacetate (4.75 g.12 mmoles) is suspended intetrahydrofuran (24 ml.) and added with triethylamine (1.68 ml.- 12mmoles) while cooling in an ice-water bath. Tert.-butyloxycarbonylnitro-L-arginine 2,4-dinitrophenyl ester (7.15 g.-14.4mmoles) is added to the clear solution and the reaction mixture is keptat room temperature. At one hour intervals, two more portions of theabove mentioned dinitrophenyl ester (0.7 g. each) are added. Afterovernight standing at room temperature, the reaction mixture is dilutedwith ethyl acetate (ca. 200 ml.) and washed once with 20% aqueous citricacid, once with water; ten. times: with .0.5 .N ammonium hydroxide andfour.:times with 'water.i'The. organic phase .is' dried over magnesiumsulfate and. the: solvent is removed in .vacuo. :Ilie :res'idueiisdisintegratedlunder ether,--filtered and dried. This crude product isboiled with ether. (ca. 80 ml.)." while adding small portions'of ethylacetate (upf'to a-jtotal.:.of ca. 20 ml.). The amorphousjso'lid slowlychanges into a crystalline mass. After overnight standing at roomtemperature, the product is filtered and washed with ethyl acetate ether(1.9). Yield 6.5 g., M.P. (120) 124-128 [M -40.2 (c. l MeOH).

Analysis.-Calc.d. for C H O N C: 51.7, H: 6.9, N: 19.3. Found: C: 51.5,H: 7.2, N: 19.2.

EXAMPLE 21 N(tertebutyloxycarbonyl-Q-benzyl L seryl-nitro-L-arginyl-L-leucyl) N benzyloxycarbonyl hydrazine (XVII)N:(tert.-butyloxycarbonylnitrorL-arginyl L leucyl)- N'-benzyloxycarbony1hydrazine (7 g.-.12 mmoles) is dissolvedin cold trifluoroacetic acid (25m1.) and the solutionis kept at room temperature for 15 minutes. Afterremoving most of the trifluoroacetic acid .in vacuo at room temperature,the residue is' disintegrated under ether, filtered, washed thoroughlywith ether, and dried in vacuo over potassium hydroxide. Thistrifiuoroacetate is dissolved in dimethylformamide (36 ml.) and addedwith'triethylamine (1.68 ml.12 mmoles)and'terL-butyloxycarbonyl-O-henzyl-L-serine p-nitrophenylester (preparedfrom 15 mmoles of tert.-butyloxycarbonyl-O- benzyl-L-serine). Thereaction mixture is'left. at room temperature overnight, diluted withethyl acetate and washed once with 20% aqueous citric acid and threetimes'with water. The organic layer is dried over magnesium sulfate andthe solvent is removed in vacu0., The residue is disintegratedunderether, filtered, washed with ether and dried. This crude product iscrystallized from ethyl acetate. Yield 6.3 g. The sample for analysis isdried overnight at 60, M.P. (110) 115-118 ['or]' -37.7 (c. l MeOH).

A n alysis.-Calcd. for C H N O C: 55.5, H: 6.8, N: 16.6. Found: C: 55.3,H: 7.2, N: 16.5,

EXAMPLE 22 i N (tert. butyloxycarbonyl L leucyl O benzyl-L- serylnitro Larginyl L leucyl) N' -.benzy1oxycarbonyl hydrazine (XVIII) N (tert.butyloxycarbony-O-benzyl-L-seryl-nitro-L-arginyl-L-leucyl-N'-benzyloxycarbonyl hydrazine (ca. 1 g:8 mmoles) isdissolved in cold trifluoroacetic acid (23 ml.) and the solution is keptat room temperature for minutes. The excess trifluoroacetic acid isremoved in vacuo and the oily residue is disintegrated with ether,filtered, 'washed thoroughly with ether and dried in vacuo o'ver KOH.This trifiuoroacetate is dissolved in dimethylforrnarrride (24ml.) andto the resulting solution, triethylamine (1.12 ml.) and'tert.-butyloxycarbonyl-L- leucine'p-nitrophenylester (3.52 g.-1Ommoles) are added. The reaction mixture is left at room temperatureovernight, diluted with ethyl acetate and washed once with citric acidand twice with water. A precipitate appearedin the organic; phaseduring,the-washings.;The organic phase with the solid in suspensionisconcentrated to dryness in vacuo and the crystalline residue isrecrystallized twice from absolute ethanol. Yield 5.8 g., M.P. 200-202ahj -21 (c. 1.4 dimethylformamide).

Analysis.-Calcd. for C4 H 2N1'0O f C: HI N :'16;1. Found: C: 55.9,H:'7.1, N: 16.1. Y i

' EXAMPLEv 23' 1 N (tert. butyloxycarbonyl v benzyl L glutamyl- -Lleucyl O benzyl L seryl nitro L arginyl- L-leucyl) -N'-benzyloxycarbonylhydrazine (XIX) I N (tert.butyloxycarbonyl-L-leucyl-O-benzyl-L-serylnitro-barginyl-L-leucyl)-N'-benzyloxycarbonylhydrazine (3.5 gL-4 mmoles) is dissolved in cold trifluoroacetic acid(15 ml.) and the solution is kept at room temperaturefor 15 minutes.Most of the trifluoroacetic acid is removed in va'cuo and the residue istriturated with ether; filtered, washed-thoroughly with ether and driedin vacuo. This trifluoroacetate is dissolved in dimethylformamide (14ml.) and to the resulting solution triethylamine (0.56 ml.) andtert.-butyloxycarbonyl-v-benzyl-L-glutamic acid p-nitrophenylester (2.3g.5 mmoles) are added. After overnight standing at room temperature, afew drops of acetic acid are added and the solvent is removed in vacuo.The solid residue is disintegrated under ethyl acetate, filtered anddried. This material is recrystallized from methanol. Yield 3.65 g.,M.P. 220-221.

AnalySis-Calcd. for C H N O C: 58.4, H: 6.93, N: 14.14. Found: C: 58.8,H: 7.13, N: 14.54.

EXAMPLE 24 (a) Benzyloxycarbonyl-nitro-L-arginyl-L-leucine methyl esterI (1) To a solution of benzyloxycarbonyl-nitro-L-arginine 1.77 g.) and2,4-dinitrophenol (0.92 g.) in tetrahydrofuran (30 ml.),dicyclohexylcarbodiimide (1.05 g.) is added and the mixture kept at roomtemperature (about 23 C.) for 45 minutes. Three drops of acetic acid areadded, the N,N-dicyclohexylurea is removed by filtration and is washed.with tetrahydrofuran (20 ml.). The filtrate and washings are combined,the solvent removed in vacuo and the residue triturated with ether. The2,4-dinitrophenyl benzyloxycarbonyl-nitro-L-arginate is filtered andwashed with ether and weighs 2.5 g., M.P. ca. 60 dec. A sharp CO band isrevealed in the LR. spectrum at 5.62 Il'lu.

To a solution of methyl L-leucinate hydrochloride (1.0 g.) in chloroform(10 ml.), triethylamine (1.0 ml.) and the above crude active ester (2.0g.) are added. Evolution of heat is observed. The solvent isconcentrated in vacuo and the residue dissolved in absolute ethanol (50ml.). Crystals separate from the solution. The product is washed withethanol (25 ml.) and ethyl acetate (10 ml.) is dried at roomtemperature. A second crop is obtained by concentration' of the motherliquor. Total yield is 1.0 g., M.P. 160-161". (2) The same protecteddipeptide ester (VII) is also prepared by the mixed anhydride methodusing pivaloyl chloride as the activating reagent-The product isrecrystallized from ethanol and is secured in 50-55% yield, M.P.161-162, [bt] 22 (c. 1 MeOH). The melting point is unchanged even afterseveral crystallizations from ethanol or acetone; prolonged drying invacuo at however, raises the melting point to 162-163".

Analy'sis.-'Calcd. for VII, C H N O- C: 52.5, H: 6.7, N: 17.5. Found: C:52.6, 'H: 6.5, N: 17.3.

(b) ,N-benzyloxycarbonyl-O-acetyl-L-seryl-nitro-L- arginyl-L-leucinemethyl ester The protected dipeptide ester from (a) (9.6 g.) issuspended in acetic acid (40 ml.) and treated with a solution ofhydrobromic acid in acetic acid (ca. 5 N, 30 ml.). Within a few minutes,a clear solutionresults. After about one hour at room temperature,ether; (700 ,ml.) is added to the solution and the semi-solid depositwhich forms is triturated with fresh. etherZThe hydrobromide isfiltered, washed with ether (600 ml.) and dried in vqcuo over sodiumhydroxide for a short time. It is dissolved in dimethylformamide (50ml.) and triethylamine (6 ml.) and N-benzyloxycarbonyl-O-acetyl-L-serinep-nitrophenylester (10 g.) are added to the mixture.

After two hours at room temperature, the precipitated triethylam'moniumbromide is filtered 011 and washed-with dimethylformamide (25 ml.).Acetic acid (5 ml.) is added to the combined filtrates whichare thendiluted with ethyl acetate (500 ml.), refiltered, and washed three timeswith 500 ml. portions of water. The water washes arere-extracted withethyl acetate (300 ml.). The ethyl acetate solutions are evaporated invacuo and the oily residue triturated with ether. The product is washedwith ether (a total of 600 ml.) and dried in air which weighs 12.2 g.,M.P. 90105. The crude product is dissolved in chloroform (100 ml); thecrystals which separate are washed with chloroform (50 ml.) and dried,weighing 8.6 g. (70% M.P. 1l5-ll9 with sintering at 113", [001 --27 (c.2 95% EtOH).

Analysis.Calcd. for VIII, C H N O C: 51.2, H: 6.5, N: 16.1. Found: C:51.3, H: 6.4, N: 16.0.

A second crop is obtained from the mother liquor weighing 1.5g. (12%),M.P. ca. 115.

(o) Tert.-butyloxycarbonyl-L-leucyl-O-acetyl-L-serylnitro-L-arginyl-L-leucine methyl ester (XX) Nbenzyloxycarbonyl-O-acetyl-L-seryl-nitro-L-arginyl- L-leucine methylester (6.2 g.-l mmoles) is dissolved in a mixture of glacial acetic acid(30 ml.) and hydrogen bromide in acetic acid (30 ml.-ca. 4 N). After onehour standing at room temperature, ether is added (450 ml.) and theprecipitate is filtered, washed thoroughly with ether and dried invacuo. This hydrobromide is dissolved in dimethylformamide (35 ml.) andto the resulting solution triethylamine (3.0 ml.) andtert.-butyloxycarbonyl L-leucine p-nitrophenylester (3.90-11 mmoles) areadded. The reaction mixture is kept at room temperature overnightdiluted with ethyl acetate (150 ml.) and washed with 20% aqueous citricacid and water. Theorganic layer is dried over magnesium sulfate and thesolvent is removed in vacuo. The crystalline residue is taken-up withethyl acetate, filtered, washed with ethyl acetate and dried. Yield 5.58g., M.P. (170) 176-178 [0:]; 27.2 (c. 1.1 dirnethylforrnamide).

AHGIYSiSr-Cfildd. for C H O N CI 50.6, H: 7.6, N: 16.3. Found: C: 50.2,H: 7.9, N: 15.9.

EXAMPLE 25 Tert. butyloxycarbonyl-'y-benzyl-L-glutamyl-L-leucyl-G acetylL acryl-nitro- Larginyl-L-leucine methyl ester Tert. butyloxycarbonyl Lleucyl-O-acetyl-L-serylnitro-L-arginyl-L-leucine methyl ester (4.1 g.-6mmoles) is dissolved in cold trifluoroacetic acid (15 m1.) and thesolution kept at room temperature for 15 minutes. Most of thetrifluoroacetic acid is removed in vacuo and the residue is trituratedwith ether, filtered, washed thoroughly with ether and dried in vacuo.This trifluoroacetate is dissolved in dimethylformamide and theresulting solution is added with triethylamine (0.84 ml.) andtert.-butyloxycarbonyL-y-benzyl-L-glutamic acid p-nitrophenylester (3.4g.-7 .5 mmoles). The reaction mixture is kept at room temperature for 4hours, diluted with ethyl acetate, washed with 20% aqueous citric acid,and water. The oryanic phase is dried over magnesium sulfate and thesolvent is removed in vacuo. The crystalline residue is taken-up inethyl acetate, filtered, washed with ethyl acetate and dried. M.P. (195)197-198 [04 23.9 (c. 1.1 dimethylformamide).

Analysis.Calcd. for C H O N C: 54.23, H: 7.21, N: 13.89. Found: C:54.62, H: 7.34, N: 13.89.

EXAMPLE 26 Tert.-butyloxycarbonyl-L-glutamyl-L-leucyl-L-seryl-Larginyl-L-leucine hydrazide (XXII) Tert. butyloxycarbonyl-y-benzyl-Lglutamyl-L-leucyl- O-acetyl L seryl-nitro-L-arginyl-L-leucine methylester (4.2 g.) is dissolved in 80% aqueous acetic acid and hydrogenatedin the presence of 10% palladium on charcoal (500 mg.) for 48 hours. Thecatalyst is filtered off and the filtrate is freeze dried. Thisprotected pentapeptide methyl ester (3.6 g.) is dissolved in methanolichydrazine (175 ml.) and the solution is kept at room tem-' perature for3 hours. After this period it is concentrated to half volume in vacuoand ethyl acetate added until complete precipitation. The precipitate isfiltered, washed EXAMPLE 27 Benzyloxycarbonyl-L-threonyl-L-serine methylester (XXIII) To a solution of 0.93 g. (6 mmoles) of serine methyl esterhydrochloride in 10 ml. of dimethylformamide, 0.84 ml. of triethylamineis added followed by 1.68 g. of benzyloxycarbonyl-L-threonine2,4-dinitrophenyl ester. The mixture is stirred for an hour at roomtemperature. The solvent is removed in vacuo, and the residue isextracted with a mixture of 60 ml. of 0.1 N hydrochloric acid and 60 ml.of ethyl acetate. The ethyl acetate solution is washed successively with3 X 50 ml. of saturatedsodium bicarbonate solution and 2X 50 ml. ofwater. Evaporation of the solvent and washing the residue with a littleether yields 0.65 g. of light yellowish white crystals, M.P. 1 3'4 136.The mother liquors are combined and extracted with 2X ml. of ethylacetate. After washing the" ethyl acetate solution with 2X 50 ml. ofwater, the solvent is removed in vacuo, and the residue washed withether to give 0.49 g. of crystals, M.P. 134136 (total yield 84%Crystallization of a sample from a mixture of benzene and methanol didnot raise the melting point; [a] -1.1 (c. 2.2 ethanol), [ah -"+630 (c.0.46 dimethylformamide).

Analysis.Calcd. for ClcHmOqNz: OCH3, 8.78; Found: 8.79. I

EXAMPLE 28 Benzyloxycarbonyl-L-phenylalanyl-L-threonyl-L serine methylester (XXIV) A solution of 10.5 g. (30'mmoles) ofbenzyloxycarbonyl-L-threonyl-L-serine ester in 100 ml. of ethylalcoholand 30 ml. of 1 N HCl is hydrogenolyzed over 1.05 g. of 10% palladium oncarbon for 7 hours. The catalyst is filtered and the solution isevaporated to a syrup.:To a solution of the unprotected dipeptide in ml.of dimethylformamide containing 4.2 ml. of triethylamine, 14.5 g. (36mmoles) of benzyloxycarbonyl-L-phnylalanine p-nitrophenylester is addedand the mixture stirred at room temperature for 6 hours. The solvent isremoved in vacuo, and the residue digested with 150 ml. of ethylacetate. The crystalline solid is collected and dried, 13.1 g. (88%yield), M.P. 168-172. An analytical sample is obtained by twocrystallizations from dilute methanol, M.P. 179-181", [ab -6.8 (c. 0.84dimethylformamide).

Analysis.-Calcd. for C H N O C: 59.78, H: 6.23, N: 8.38. Found: C:59.72, H: 6.14, N: 8.30.

EXAMPLE 29 Benzyloxycarbonyl-L-threonyl-L-phenylalanyl-L-threonyl-L-serine methyl ester (XXV) (a) By the azide procedure: Toasolution of 3.32 g. (8 mmoles) ofbenzyloxycarbonyl-L-threonyl-L-phenylalanine hydrazide in a mixture of10 ml. of dimethylformamide and 5 ml. of concentrated hydrochloric acidcooled to --15", a solution of 0.56 g. of sodium nitrite in 2 ml. ofwater is added during the course of 3 minutes. The reaction mixture isstirred at 10 for 10 minutes, and the pH adjusted to 6.5 with ca. 6.9ml. of triethylamine. To the above mixture, a solution of L-threonyl-L-serine methyl ester hydrochloride [obtained by the hydrogenolysis of.2.83 g. (8 mmoles) of benzyloxycarbonyl L- threonyl-L-serine methylester in ethanol over Pd/C] in ml. of dimethylformamide containing 1.1ml. of triethylamine' is added. The mixture is stirred'at --10 forminutes, and then left overnight at 4. The solvent is removed in vacuo,and the solid that separated upon addition of ml. ofwater is collected,washed with a mixture of ethyl acetate and ether, and dried to'give 1.45g. of a crude product, M.P. 142152. Crystallization from dilute methanolgives 0.71 g. (15% yield) of white crystals, M.P. l84-l87 [a] 4.0 (c.0.53 dimethylformamide).

Analysis.Calcd. for C H N O C: 57.79, H: 6.38, N: 9.30. Found: C: 57.25,H: 6.67, N: 9.38.

(b) By the active ester method: A solution of 5.01 g. (10 mmoles) ofbenzyloxycarbonyl-L-phenylalanyl-L- threonyl-L-serine methyl ester in500 ml. of ethyl alcohol and 10 ml. of l N HCl is hydrogenolyzed over0.5 g. of 10% palladium on carbon for 8 hours. After filtering ofl. thecatalyst, the solvent is removed in vacuo to give a pale yellow solid.To a solution of the solid in 100 ml. of dimethylformamide containing1.4 ml. of triethylamine, 4.98 g. (12 mmoles) ofbenzyloxycarbonyl-L-threonine dinitrophenyl ester is added, and thesolution stirred overnight at, room temperature. After removing thesolvent in vacuo, 300 ml. of ethyl acetate is added and the solid thatseparated is collected and crystallized from dilute methanol to give 5.8g. (96% yield) of white crystals,

M.P. 190-.l 92. An analytical sample is obtained by two crystallizationsfrom dilute methanol, M.P. 19 -196,

[a] 9.2 (c. 0.6 dimethylformamide).

Analysis. Calcd. for C29H38N4O11: C: 57.79, H: 6.38,

" N: 9.30. Found; c; 57.58, H: 6.58, N: 9.24.

EXAMPLE 30 Benzyloxycarblonyl L-threonyl-L-phenylalanyl-L-threonyl-L-serine hydrazide (XXVI) To a solution of 4.2 g. (7 mmoles) ofbenzyloxycarbonyl L threonyl-L-phenylalanyl-L-threonyl-L-serine .methylester in 120 ml. of methyl alcohol, 3 ml. of hydra- EXAMPLE 31,BBenzyl-L-aspartylglycine trifiuoroacetate (XXIX) A solution of glycine(2.65 g., 35.3 mmoles) in a mixture of water (45 ml.) and pyridine (45m1.) is adjusted to pH 8.5 with 4 N NaOH. The p-nitrophenyl ester oft-butyloxycarbonyl-B-benzyl-L-aspartic acid (14.70 g., 33.1 -mmoles) isgradually added as the powdered solid over a 2 /2 hour period while thepH is kept in the range of 8.3

to 8.5 byaddition of 4 N sodium hydroxide (in total 14.7 ml., 588milliequivalents). After stirring for an additional hour, the'clearsolution is diluted with 100 ml. of water, adjusted to pH 6.1 andextracted 3 times with equal volumes of ether-hexane (60:40). Theaqueous layer is then cooled, adjusted to pH 3 and extracted four times.with ethyl acetate. The organic phase is washed once with water,'driedover sodium sulfate and concentrated to dryness. The amorphous residue(10 g., 80% yield) does not crystallize, but can 'be converted to acrystalline dicyclohexylamine salt which is recrystallized from acetone(starts to melt at 126, then resolidifies and finally melts at190-200"). Reconversion to the free acid by extraction with ethylacetate from 20% aqueous citric acid still does not yield a crystallinematerial. The amorphous t-butyloxy-carbonyl fl benzyl-L-aspartylglycine(9.7 g., 24.6 mmoles) is dissolved in trifluoroacetic acid 15 minutes.The major part of the trifluoroacetic acid is then removed byevaporation in vacuo, and dry ether (200 ml.). is added to the residue.After cooling and triturating the mixture, the solid which formed iscollected by filtration, washed three times with 'cold ether and driedto yield 8.4 g. of product (M.P. 127132). Paper chromatography (solventsystem I, butanol-acetic acidpyridine-water, 3026;20:24) reveals twoninhydrin positive spots (R 0.26, 0.63). The brownish spot (R, 0.63) isthe major one. The material is recrystallized by dissolving in water(260 ml.) at 40 and cooling. The solid is filtered, washed and dried toyield 4.4 g. of crystals (M.P. 138-143 This material produces only oneninhydrin positive spot (R 0.63, solvent system I), on paperchromatograms. The NMR spectra (deuterated dimethylsulfoxide), 2.62-1-(5H, aromatic), 4.7% (2H, benzyl methylene), 5.757 (triplet, 1H, aprotonin aspartic), 6.137 (d, 2H, J=6 cps.), 7.061 (d, 2H, J=6 cps., aspartylmethylene), agreed with the desired structure, fl-be'nzylaspartylglycine. The fluorine analysis (13.33% F found) indicated theproduct is the trifluoroacetate salt.

EXAMPLE 32Benzyloxycarbonyl-L-histidyl-Lseryl-fi-benzyl-L-aspartylglycine (XXX) to0 and brought into solution by the addition of concentrated hydrochloricacid (7.0 ml., 84.0 mmoles). While stirring and cooling this solution to10, a cold aqueous solution (1.66 ml.) containing 0.906 g. (13.13mmoles) of sodium nitrite is gradually added during a 3 minute period.The mixture is stirred for an additional 6 minutes at 10. To thissolution of the azide is added a cold solution of the trifluoroacetatesalt of fi-benzyl-L-aspartylglycine (5.25 g., 13.3 mmoles) in thedimethylformamide (23 ml.). Additional dimethylformamide (2 ml.) is usedto wash in any of the amino component left behind. After cooling thereaction mixture to 20, cold triethylamine (10.83 g., 107 mmoles) isadded to bring the pH to 7. This neutralization causes -a gelatinousprecipitate to appear and the heterogeneous mixture is stirred at 10 for20 minutes and then at 5 for 96 hours. The precipitate is filtered 011and washed with cold dimethylformamide (25 ml.). The filtrate and washesare concentrated in vacuo with a bath temperature not higher than 35.Chloroform is added to give a final volume of 2.50 ml. After cooling themixture, the white precipitate which had formed is filtered off andwashed with cold chloroform. This solid is then triturated with coldether, filtered and washed three times with more cold ether. When dried,the solid weighs 8.80 g. On paper chromatograms (butanol-aceticacid-pyridine-water, 30:6:20z24, solvent system I) four spots (R 0.81,0.69, 0.64 and 0.54) are produced by Pauly reagent. There are noninhydrin positive spots. In preparation for a oountercurrent separation(using butanol-pyridine-acetic acid-water, 4:2:1:7), the product isdissolved in lower phase (180 ml.). However, addition of an equal volumeof upper phase caused a. crystalline material to appear. After themixture is cooled, the product is filtered, washed and dried (4.15 g.,50%, M.P. dried in vacuo at 30 over potassium hydroxide) and found to behomogeneous (R 0.81) in the paper chromatograms (solvent system I, Paulyreagent spray) and on silica gel plates (R 0.75; 'butanolaceticacidwater). The material in the mother liquor is then separated bycountercurrent distribution (475 transfers) to give four peaks (K values2.47, 1.0, 0.74, 0.51). Each peak tube is chromatographicallyhomogeneous (paper chromatographic system described and Pauly reagentspray) and the R values are respectively 0.82, 0.70, 0.63

and 0.54. From one peak (K 2.47), crystalline material .the followingratio of amino acids: aspartic:serine:gly-

cinezhistidine, 1.0:0.82:0.93: 1.14.

EXAMPLE 3 3 A small portion of the crystalline product (32 mg.)dissolved in methanol-acetic acid-water (:1:4) ml.) is hydrogenolyzed inthe presence of 10% palladium on charcoal (7 mg.) for 3 hours atatmospheric pressure. The product when recrystallized from ethanol-Wateryields 16 mg. of the free tetrapeptide (M.P. 283; dec.). This materialproduced only one ninhydrin positive spot (R; 0.10) on paperchromatograms (solvent system I). The protected tetrapeptide is solublein dimethylformamide, pyridine and methanol and is recrystallized bydissolving in a minimum amount of pyridine without heating, adding asmall amount of water and cooling. The white crystals melt at 12813l.

EXAMPLE 34 L glutamyl L leucyl L-seryl-L-arginyl-L-leucyl-L- arginyl Laspartyl L seryl-L-alanyl-L-arginyl-L leucyl L glutaminylL-arginyl-L-leucyl-L-leucyl-L- glutaminylglycyl-L-leucyl-L-valinamide(XXXI) (a) Tert. butyloxycarbonyl L-glutamyl-L-leucyl-L- seryl L arginylL leccyl-nitroL-arginyl-fi-benzyl-L- aspartyl Obenzyl-L-seryl-L-alanyl-nitro-L-arginyl-L- leueyl L glutaminylnitro-L-arginyl-L-leucyl-L-leucyl- L glutaminylglycylL-leucyl-L-valinamide (XXXIa): Tert. butyloxycarbonyl Lglutamyl-L-leucyl-Irseryl- L-arginyl-L-leucine hydrazide (450 mg.-0.6mmole) is dissolved in dimethylformamide (4.5 ml.) and the solution iscooled in a Dry Ice-acetone bath at 20. Concentrated hydrochloric acid(0.3 ml.) and 14% aqueous sodium nitrite are added (0.45 ml.), and theresulting solution is stirred in the cooling bath at -15 for 5 minutes.After this period the temperature of the cooling bath is lowered to andthe reaction mixture is added with N-ethyl piperidine (0.42 ml.)followed by a solution of nitro Larginyl-fl-benzyl-L-aspartyl-O-benzyl-L- seryl L alanylnitro-L-arginyl-L-leucyl-L-glutaminylnitro Larginyl-L-leucyl-L-leucyl-L-glutaminylglycyl- L-leucyl-L-valinamidetrifluoroacetate (XIVa) (873 mg.) in dimethylformamide (4.5 ml.). Themixture is kept in the cooling bath for 10 minutes and then stored at 05for three days. The solvents are removed in vacuo and the residue isdisintegrated under ethyl acetate, filtered and dried.

(b) L glutamyl L leucyl-L-seryl=L-arginyl-L-leucylnitro L arginylS-benzyl-L-aspartyl-O-benzyl-L-seryl- L alanyl nitroL-arginyl-L-leucyl-L-glutaminyl-nitro- L arginyl Lleucyl-L-leucyl-L-glutaminylglycyl-L- leucyl-L-valinamide (XXXIb): Thisproduct is dissolved in cold trifiuoroacetic acid (10 ml.) and thesolution is kept at room temperature for 15 minutes. Ether is addeduntil complete precipitation and the precipitate is filtered, washedwith ether and dried. This material is suspended in water (10 ml.),thoroughly disintegrated, centrifuged down, resuspended in water (10ml.), centrifuged down and dried in vacuo. Yield 800 mg. This materialshows on paper chromatography only one spot which is UV absorbing andSakaguchi positive.

(c) L glutamyl L leucyl-L-seryl-L-arginyl-L-leucyl- L arginyl L aspartylL seryl-Lalanyl-L-arginyl-L- leucyl Lglutaminyl-L-arginyl-Lleucyl-L-leucyl-L-glutaminylglycyl Lleucyl-L-valinamide: A small sample of the product from (b) ishydrogenated for 48 hours in the presence of 10% palladium on charcoal.The product thus obtained (XXXI) is shown to be identical to thenonadecapeptide prepared by the stepwise procedure.

20 EXAMPLE 35 L threonyl L phenylalanyl L threonyl-L-seryl-L- glutamyl Lleucyl L-seryl-L-arginyl-L-leucyl-L- arginyl L aspartyl L serylL-alanyl-L-arginyl-L- leucyl L glutaminyl L-arginy1-L-leucyl-L-leucyl-L-glutaminylglycyl L leucyl L valinamide acetate (XXXII) BenzyloxycarbonylL threonly L-phenylalanyl-L- threonyl-L-serine hydrazide (12.0 mg.0.2mmoles) is dissolved in dimethylformamide (1.5 ml.) and the solutioncooled in a Dry Ice-acetone bath at -20 and added with concentratedhydrochloric acid (0.1 ml.) and 14% aqueous sodium nitrite (0.11 ml.)After 5 minutes at 15 C. the temperature of the bath is brought down to25 and N-ethyl piperidine (0.14 ml.) is added followed by a. solution ofthe partially protected nonadecapeptide amide trifiuoroacetate. Thereaction mixture is stored for two days at 0 3", added with anotherportion of tetrapeptide azide (prepared from 60 mg. of the tetrapeptidehydrazide) and kept for another two days at 0-3. The reaction mixture isconcentrated to dryness in vacuo, the residue, benzyloxycarbonyl Lthreonyl-L-phenylalanyl- L threonyl L seryl L-glutamylL-leucyl-L-seryl-L- arginyl I leucylnitro-L-arginyl-fi-benzyl-L-aspartyl- O benzyl Lseryl-L-alanyl-nitro-L-arginyl-L-leucyl-L- glutaminyl nitro L arginylIrleucyl-L-leucyl-L-glutaminylglycyl L leucyl-L-valinamide (XXXiIa), istriturated with ethyl acetate, filtered and dried. This product ishydrogenated for 48 hours over 10% palladium on charcoal to yield thetricosapeptide amide acetate (XXXH).

EXAMPLE 36 Benzyloxycarbonyl L histidyl L seryl-'y-benzyl-L-aspartyl-glycyl L theonyl-L-phenylalanyl-L-threonyl- L seryl Lglutamyl-L-leucyl-L-seryl-L-arginyl-L leucyl L arginyl Laspartyl-L-seryl-L-alanyl-L arginyl Lleucyl-L-glutaminyl-L-arginyl-L-lucyl-L- leucyl L glutaminylglycylL-leucyl-L-valinamide (XXXIII) Benzyloxycarbonyl L histidylL-seryl-y-benzyl-L- aspartyl-glycine mg.0.2 mmoles) is dissolved inacetonitrile (1.5 ml.) and added with triethylamine (0.03 ml.) and Nethyl 5-phenylisoxazolium-3'-sulfonate (55 mg.0.2 mmoles). After 30minutes a solution of .400 mg. of the tricosapeptide amide acetate.(XXXII) in dimethylformamide (1.5 ml.) and water (0.5 ml.) are added.The reaction mixture is kept overnight at room temperature, the solventsare removed in vacuo and the residue triturated Wat absolute ethanol,filtered and, dried. This material (XXXIII), after 16 hourshydrogenation over 10% palladium on charcoal, yields secretin.

EXAMPLE 37 N benzyloxycarbonyl L histidyl-L-seryl-fl-benzyl-L-aspartylglycyl L threonyl-L-phenylalanyl-L-threonyl- O benzyl L seryl-'-benzyl-L-glutamyl-L-leucyl-O- benzyl L seryl nitroL-arginyl-L-leucyl-nitro-L- arginyl B benzyl L-aspartyl-O-benZyl-Lseryl-L- alanyl nitro L arginyl-L-leucyl-L-glutaminyI-nitro- L arginyl Lleucyl-L-leucyl-L-glutaminylglycyl-L leucyl-L-valinamide (XXXIV) Asample (163 mg.) of the partially protected tricosapeptidetrifiuoroacetate (as prepared in Examples 1 to 28 of copending U.S.application of Miklos Bodanszky, Ser. No. 550,956, now U.S. Pat.3,417,072, filed May 18, 1966) is dissolved in methanol (50 ml.) andtreated with an ion exchange resin (Amberlite XN-1003, 3 g., in OHcycle) for one hour. After removal of the resin by filtration and of thesolvent by evaporation, the residue is dissolved in dimethylformamide (2ml.). The protected tetrapeptide benzyloxycarbonylL-histidyl-L-seryl-p-benzyl-L- aspartylglycine (64 mg.) is added to thesolution followed by dicyclohexylcarbodiimide (12 mg). After one hour atroom temperature the solvent is removed in vacuo and the residuetriturated with ethyl acetate to give a solid material (XXXIV), which inturn, is dissolved in acetic acid (10 ml.) and is hydrogenated in thepresence of a 10% palladium on charcoal catalyst for 2 /2 days. Afterremoval of the catalyst by filtration, the acetic acid is evaporatedfrom the frozen state. The residue is dissolved in water. This solutionshows the characteristic hormonal properties of secretin.

EXAM PIE 3 8 Benzyloxycarbonyl L threonyl L phenylalanyl-L- threonyl Lseryl L glutamyl-L-leucyl-L-seryl-L- arginyl-L-leucine hydrazide (XXXV)(a) L glutamyl L leucyl O acetyl-L-seryl-L- arginyl L leucine methylester trifluoroacetate (XXIa): Tert.butyloxycarbonyl-v-benzyl-L-glutamyl-L-leucyl-O- acetyl Lseryl-nitro-L-arginyl-L-leucine methyl ester (XXI) (4.2 g.) is dissolvedin 80% aqueous acetic acid and hydrogenated in the presence of 10%palladium on charcoal (500 mg.) for 48 hours. The catalyst is filtered013? and the filtrate is freeze dried. The residue is dissolved in coldtrifluoroacetic acid (15 ml.). The solution is kept at room temperaturefor 15 minutes, the excess trifluoroacetic acid is removed in vacuo andthe residue triturated with ether, filtered and dried.

(b) Benzyloxycarbonyl L threonyl-L-phenylalanyl- L threonyl Lseryl-L-glutamyl-L-leucyl-L-seryl-L- arginyl-L-leucine hydrazide (XXXV):Benzyloxycarbonyl L threonyl-L-phenylalanyl-L-threonyl-L-serinehydrazide (XXXVI) (120 mg, 0.2 mmoles) is dissolved in dimethylformamide(1.5 ml.) and the solution cooled in a Dry Ice-acetone bath at 20 C. Tothe cooled solution concentrated hydrochloric acid (0.1 ml.) and 14%aqueous sodium nitrite (0.11 ml.) are added. After 5 minutes at 15 C.,the temperature of the bath is lowered to 25 C. and N-ethyl piperidine(0.14 ml.) is added, followed by a solution of L-glutamyl-L-leucyl-O-acetyl-L-seryl-L-arginyl-L-lencine methyl ester trifluoroacetate (140mg.) in dimethylformamide (1 ml.) and N- ethyl piperidine (0.03 ml.).The reaction mixture is kept at 3 C. for 3 days and diluted with ethylacetate (50 ml.). The precipitated protected nonapeptide methyl ester isfiltered and dried. This product is dissolved in methanolic hydrazineand the solution kept at room temperature for 3 hours. The solvent isconcentrated to half in vacuo and ethyl acetate is added until completeprecipitation. The protected nonapeptide hydrazine is filtered and driedin vacuo over phosphorous pentoxide.

What is claimed is:

1. A compound of the formula and physiologically acceptable acidaddition salts thereof and carboxyl activated derivatives thereofwherein R is hydrogen or an N-terminal amino protecting group, N-terminal protected L-histidyl-L-seryl wherein the N-terminal protectinggroups are benzyloxycarbonyl, t-butyloxycarbonyl, phthalyl,o-nitrosulfenyl or tosyl, and R is a fi-carboxyl protecting groupselected from the group consisting of lower alkyl or benzyl, providedthat when R is benzyloxycarbonyl, R is not methyl.

2. A compound according to claim 1 having the namebenzyloxycarbonyl-L-histidyl-L-seryl-p-benzyl-L-aspartylglycine.

3. A compound according to claim 1 having the namet-butyloxycarbonyl-fl-benzyl-L-aspartylglycine.

4. A compound according to claim 1 having the name t butyloxycarbouylp-benzyl-L-aspartylglycine dicyclohexylamine salt.

5. A compound according to claim 1 having the namefi-benzyl-L-aspartylglycine trifluoroacetate.

References Cited UNITED STATES PATENTS 3,400,118 9/1968 Bodanszky et al.260112.5 3,250,758 5/1966 Weygand et al. 260-1125 3,341,510 9/1967Chillemi 260112.5 3,352,844 11/1967 Boissonnas et al. 260112.5 3,417,07212/ 1968 Bodanszky 260-1125 OTHER REFERENCES Ondetti et al.:Pharmacology of Hormonal Polypeptides and Proteins, Bach et al. eds.,Plenum Press, New York (1968), pp. 18-31.

Schroder et al.: The Peptides, vol. I, Academic Press, New York (1965),pp. 69-75.

Klieger et al.: Ann. 640, 157-167 (1961).

Le Quesne et al.: J. Chem. Soc. 1952, 24-26.

Ondetti et al.: J. Amer. Chem. Soc. 90, 4711-4716 (1968).

Zervas et al.: J. Amer. Chem. Soc. 85, 3660-3666 (1963).

ELBERT L. ROBERTS, Primary Examiner mg? UNITED STATES PATENT OFFICECERTIFICATE OF CORRECTION Patent No. 3,812, 092 v o Dated May 21, 1974In e Miklos Bodanszky etal.

It is certified that error appears in the above-identified patent E andthat said Letters Patent are hereby corrected as shown below:

Column 1, line 25, "aspartglyglycine" should read --asparty1- g1ycine.Column 1, line' '32, insert at the end of line, -leucy1-L- Column 2,line 35, after "negative" insert Column 2, line 36-, "ester" should read-esters. Column 3, line 53, "0.5" should read -0.05-. Column 5, line30", before "1.0" insert -Val,. Column 7, line l4, "C should read CColumn 8, line 42, at the end of line, delete "L-". Column 8, line .43,at the beginning of line, delete "leucyl"-. Column 8, 1ine59, "(R -0.6)"should read -(R =0.6).

Column 8, line 64, "is" should read -'-in-.

Column 11, line 6, (XXII) should read (XIII)- Column 15, line 40,"aeryl" should read -seryl-. Column 15, line 54, "oryanic" should readorganic--. v I Column 17, line 47 should read --Analysis. Calc'd. for 1C H N O C: 55.80, H: 6.36,

Column 18, line 19, "aproton" should read -a proton. Column 19, line 30,"leccyl" should read -leucyl-. Column 19, line 30, "nitroL" should read-nitro-L-. Column 19, line 69, "Lalanyl" should read -L-a1anyl--. Column19, line- 70, "Lleucyl" should read -Lleucyl--. Column 20, line 9,"threonly" should read threonyl-. Column 20, line 38, "lucyl" shouldread -leucyl-.

Column 22, line '9 should read hydrogen, an Neterminal amino protectinggroup or N- Signed and sealed this 1st day of October 1974.

(SEAL) Attest:

MCCOY M. GIBSON JR. C. MARSHALL DANN Attesting Officer Commissioner ofPatents

